48 hours after addition of rapamycin(150 nM), the cells were imaged as described in Figure A to detect formation of punctuated GFP-LC3 structure. Quantification represented the ratio of GFP-LC3 punctuated positive cell to the total cell counted(Figure B).

H4-LC3-GFP cells were treated with 1 nM IFNA2 for the indicated periods in the presence of 200 nM rapamycin. Images of the cells were collected using an ArrayScan HCS 4.0 Reader. Representative cells are shown. The average spot intensity in 500 cells from each indicated sample was determined. Data are displayed as means ?SD of the spot intensity per cell (below). RLU, relative leight unit.

Rheb Induces Phospho-eIF2a Independent of mTORC1 and Promotes Phospho-eIF2a Predominantly through PERK (A) Western blotting of indicated proteins of HEK293 expressing cDNAs of myc (control) or myc-Rheb WT were grown in DMEM with serum (+) or without serum (-) or pretreated with the inhibitors rapamycin (250 nM) or DMSO (0.5%, control). (B) Western blotting of indicated proteins in HEK293 cells expressing myc or myc-Rheb and pretreated with rapamycin or DMSO as in (A). (C) Western blotting of indicated proteins in HEK293 cells expressing myc or myc-Rheb and pretreated with inhibitors of MAPK (PD98059, 50 mM) or PI3K (wortmannin, 100 nM), rapamycin, or DMSO.

Autophagy induced by PL-0N and PL may restrict LPS-induced inflammatory responses in RAW264.7 cells. LPS induced autophagy in RAW264.7 cells. Cells were incubated with 1 mM 3-MA or 10 uM Rapamycin in the absence or presence of LPS (1 ug/mL) for 16 h. Then immunofluorescence for LC3 was visualized using a Zeiss LSM 710 confocal microscope.

Inhibition of mTOR activity may be responsible for sorafenib-induced down-regulation of survivin. H1299 cells were treated with the indicated concentration of RAD001 or Rapamycin for 48 h. Then H1299 cells were incubated with or without 5 μM sorafenib, with or without 5 μM RAD001, and with or without 2 μM rapamycin for 48 h. The indicated protein levels were determined by Western blot analysis. β-Actin protein levels were measured as loading controls.

Immunophilins participate in SOCE activation by CN-dependent but also CN-independent signaling pathways. Fura-2 loaded platelets were suspended in HBS and subsequently incubated at 37oC for 30 min with rapamycin (500 nM). Once incubation time was over, platelets were stimulated with TG (200 nM) in a calcium free-HBS (EGTA 100 μM was added as indicated the by arrowhead) and 4 min later CaCl2 (300 µM) was added to visualized calcium entry. Changes in fura-2 fluorescence were monitored using the 340/380nm ratio and calibrated in terms of [Ca2+]c . Traces are representative of four to six independent experiments. *,**, *** represents p < 0.05, p < 0.01 and p <0.001, respect control platelets.

Rapamycin inhibits growth-dependent TCTP induction. Cells were serum-starved for 24h and restimulated with 20% FBS for the indicated times in the presence or absence of rapamycin. The graph shows the relative TCTP signal, corrected for the loading control.

2011 Dr.Ulrich Bommer of University of Wollongong. Rapamycin (Sirolimus) purchased from Selleck

Quality Control & MSDS

Related Compound Libraries

Rapamycin (Sirolimus) is available in the following compound libraries:

Research Area

Combination Therapy

Rapamycin in combination of LY294002, significantly inhibits cell viability of Rapamycin-sensitive U87-MG and T98G, more potently than each treatment alone. The combination sensitizes Rapamycin-resistant U373-MG cells to Rapamycin by stimulating the induction of autophagy but not apoptosis through inhibition of the Akt pathway, which can be augmented by the expression of dominant-negative Akt, and decreased by the expression of active Akt. [6]

Biological Activity

Rapamycin inhibits endogenous mTOR activity in HEK293 cells with IC50 of ~0.1 nM, more potently than iRap and AP21967 with IC50 of ~5 nM and ~10 nM, respectively. [1] In Saccharomyces cerevisiae, Rapamycin treatment induces a severe G1/S cell cycle arrest and inhibition of translation initiation to levels below 20% of control. [2] Rapamycin significantly inhibits the cell viability of T98G and U87-MG in a dose-dependent manner with IC50 of 2 nM and 1 μM, respectively, while displaying little activity against U373-MG cells with IC50 of >25 μM despite the similar extent of the inhibition of mTOR signaling. Rapamycin (100 nM) induces G1 arrest and autophagy but not apoptosis in Rapamycin-sensitive U87-MG and T98G cells by inhibiting the function of mTOR. [3]

Treatment with Rapamycin in vivo specifically blocks targets known to be downstream of mTOR such as the phosphorylation and activation of p70S6K and the release of inhibition of eIF4E by PHAS-1/4E-BP1, leading to complete blockage of the hypertrophic increases in plantaris muscle weight and fibre size. [4] Short-term Rapamycin treatment, even at the lowest dose of 0.16 mg/kg, produces profound inhibition of p70S6K activity, which correlates with increased tumor cell death and necrosis of the Eker renal tumors. [5] Rapamycin inhibits metastatic tumor growth and angiogenesis in CT-26 xenograft models by reducing the production of VEGF and blockage of VEGF-induced endothelial cell signaling. [6] Rapamycin treatment at 4 mg/kg/day significantly reduces tumor growth of C6 xenografts, and tumor vascular permeability. [7]

Features

Protocol(Only for Reference)

Kinase Assay:

HEK293 cells are plated at 2-2.5×105 cells/well of a 12-well plate and serum-starved for 24 hours in DMEM. Cells are treated with increasing concentrations of Rapamycin (0.05-50 nM) for 15 minutes at 37 °C. Serum is added to a final concentration of 20% for 30 minutes at 37 °C. Cells are lysed, and cell lysates are separated by SDS-PAGE. Resolved proteins are transferred to a polyvinylidene difluoride membrane and immunoblotted with a phosphospecific primary antibody against Thr-389 of p70 S6 kinase. Data are analyzed using ImageQuant and KaleidaGr

Cell Assay:

Cells are exposed to various concentrations of Rapamycin for 72 hours. For the assessment of cell viability, cells are collected by trypsinization, stained with trypan blue, and the viable cells in each well are counted. For the determination of cell cycle, cells are trypsinized, fixed with 70% ethanol, and stained with propidium iodide using a flow cytometry reagent set. Samples are analyzed for DNA content using a FACScan flow cytometer and CellQuest software. For apoptosis detection, cells are stained with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique using an ApopTag apoptosis detection kit. To detect the development of acidic vesicular organelles (AVO), cells are stained with acridine orange (1 μg/mL) for 15 minutes, and examined under a fluorescence microscope. To quantify the development of AVOs, cells are stained with acridine orange (1 μg/mL) for 15 minutes, removed from the plate with trypsin-EDTA, and analyzed using the FACScan flow cytometer and CellQuest software. To analyze the autophagic process, cells are incubated for 10 minutes with 0.05 mM monodansylcadaverine at 37 °C and are then observed under a fluorescence microscope.

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species

Mouse

Rat

Rabbit

Guinea pig

Hamster

Dog

Weight (kg)

0.02

0.15

1.8

0.4

0.08

10

Body Surface Area (m2)

0.007

0.025

0.15

0.05

0.02

0.5

Km factor

3

6

12

8

5

20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by

Animal B Km

Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Center for International Blood and Marrow Transplant Rese
...moreCenter for International Blood and Marrow Transplant Research|National Marrow Donor Program|Resource for Clinical Investigation in Blood and Marrow Transplantation

* <1 mg/ml means slightly soluble or insoluble.* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

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LY294002 is the first synthetic molecule known to inhibit PI3Kα/δ/β with IC50 of 0.5 μM/0.57 μM/0.97 μM in cell-free assays, respectively; more stable in solution than Wortmannin, and also blocks autophagosome formation.

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Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.